The present invention relates to steroid hormone binding proteins, genes thereof, and production and use of the proteins and the genes.
It is generally thought that steroid hormones exert their physiological influence by regulating transcriptional activities. Very recently, however, steroids that exhibit their activities rapidly without acting on genes have become widely known, but this cannot be explained by the above theory. Evidence of this rapid action of steroids has been shown for every steroid in many species and tissues. Examples include the rapid action of aldosterone on lymphocytes and vascular smooth muscle (Wehling, M. (1995) Cardiovasc. Res. 29(2), 167-171), vitamin D3 on epithelial cells, progesterone on sperm (Revelli, A.; Modotti, M.; Piffaretti-Yanez, A.; Massobrio, M.; and Balerna, M. (1994) Hum Reprod 9 (5), 760-766), neurosteroids on neurons, and estrogen on blood vessels. The signal recognition and transduction mechanisms of these activities are currently being studied. As a result, it is now becoming clear that the signal recognition and transduction system resembles cascade systems of membrane receptors and the second messengers, such as those of catecholamines and peptide hormones, since many of the activities depend on phospholipase C, phosphoinositide turnover, intracellular pH, intracellular calcium, protein kinase C, tyrosine kinases, etc., (Baran, D. T. (1994) J Cell Biochem 56 (3), 303-306; de Boland, A. R. and Nemere, I. (1992) J Cell Biochem 49 (1), 32-36). Although the physiological or pathological relevance is not clear, it has been presumed that the rapid action of steroids can also be observed in vivo in the cardiovascular system, the central nervous system, and the reproductive system. It was expected that these receptors would be cloned soon and that the relationship between the rapid action of steroids and their clinical effects would be clarified (Wehling, M. (1997) Annu Rev Physiol 59, 365-393; and Wehling, M. (1995) J Mol Med 73 (9), 439-447).
In recent years, the progesterone membrane binding protein (PMBP), a membrane binding type that differs from usual steroid hormone receptors of the intranuclear transcription regulation type, was finally cloned for the first time from a pig (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89). This protein was purified from the microsome fraction, has a hydrophobic region near its N terminus, and shows no homology to existing steroid receptors. To date, a putative human homologue of PMBP, the xe2x80x9cputative progesterone binding protein genexe2x80x9d (LOCUS, HSPROGBIN; accession number, Acc.Y12711), and a putative rat homologue, xe2x80x9c25Dxxe2x80x9d (LOCUS, RNU63315; accession number, Acc.U63315), have been isolated. The pig PMBP has been well characterized, and it has been reported to bind not only to progesterone but also to corticosterone, cortisol, promegestone, and testosterone (Meyer C. (1996) Eur. J. Biochem. 239, 726-731).
The discovery of these membrane-bound steroid hormone binding proteins suggests the existence of a mechanism in the organism for regulating hormone action that differs from the one for the receptors involved in the intranuclear transcription regulation. Therefore, it should be possible to develop novel drugs that distinguish the affinities or biological activities of the membrane-bound steroid hormone binding proteins from those of the intranuclear transcription regulation type receptors using the membrane-bound steroid hormone binding proteins.
An objective of the present invention is to provide a novel steroid hormone binding protein having homology to PMBP and its gene, and also methods for producing it and uses thereof.
In order to achieve the above objective, the present inventors discovered ESTs that are inferred to be parts of a cDNA encoding a protein having homology to PMBP (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89), which is a membrane-bound steroid hormone binding protein. The present inventors then extracted a consensus sequence from the sequence information of the ESTs and performed a polymerase chain reaction on human genes using primers designed according to the consensus sequence. As a result, they have succeeded for the first time in isolating the gene encoding a novel steroid hormone binding protein having homology to PMBP from a human.
The present invention relates to a novel steroid hormone binding protein having homology to PMBP and its gene, and also methods for producing it and uses thereof. More specifically, it relates to:
(1) a protein comprising the amino acid sequence of SEQ ID NO:4,
(2) a protein having a steroid hormone-binding activity, comprising the amino acid sequence of SEQ ID NO:4 wherein one or more amino acids are substituted, deleted, and/or added,
(3) a DNA encoding the protein of (1) or (2),
(4) a vector carrying the DNA of (3),
(5) a transformant expressibly retaining the DNA of (4),
(6) a method for producing the protein of (1) or (2), the method comprising culturing the transformant of (5),
(7) an antibody that binds to the protein of (1),
(8) a method for screening a compound that binds to the protein of (1) or (2), the method comprising selecting a compound that binds to the protein of (1) or (2) by contacting a test sample with the protein of (1) or (2),
(9) a compound that binds to the protein of (1),
(10) the compound of (9) isolable by the method of (8),
(11) a method for screening a compound that specifically binds to the protein of (1) or (2) or a steroid hormone receptor of the intranuclear transcription regulation type, the method comprising selecting a compound that specifically binds to the protein of (1) or (2) or the steroid hormone receptor of the intranuclear transcription regulation type by contacting a test sample with the protein of (1) or (2) and the steroid hormone receptor of the intranuclear transcription regulation type,
(12) a compound that specifically binds to either the protein of (1) or (2) or the steroid hormone receptor of the intranuclear transcription regulation type,
(13) the compound of (12) isolable by the method of (11), and
(14) a DNA comprising at least 15 nucleotides, which specifically hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO:3.
The present invention also relates to a protein of human origin, xe2x80x9chSMBP2,xe2x80x9d which has homology to the pig membrane-bound progesterone binding protein (progesterone membrane binding protein (PMBP)) (Falkenstein, E.; Meyer, C.; Eisen, C.; Scriba, P. C.; and Wehling, M. (1996) Biochem Biophys Res Commun 229 (1), 86-89). The nucleotide sequence of the hSMBP2 cDNA isolated by the present inventors is shown in SEQ ID NO:3, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO:4.
FIG. 1 compares the amino acid sequence of hSMBP2 and those of proteins having homology to it, pig PMBP, rat 25Dx, and hSMBP1. hSMBP1, like hSMBP2, is a human gene isolated by polymerase chain reaction (PCR) using primers designed according to consensus sequences extracted from multiple ESTs having homology to pig PMBP. A gene identical to hSMBP1 has been registered in a database (Acc. Y12711). As apparent from FIG. 1, although hSMBP2 has significant homology to pig PMBP, it has a somewhat lower degree of homology except near its C terminus, unlike hSMBP1, which has a high degree of homology as a whole. hSMBP2 is thus presumed to be a protein belonging to the same family but having a different spectrum of steroids to bind.
The steroid hormone binding protein of the present invention is thought to be a membrane-bound type, which differs from the usual intranuclear transcription regulation receptors. This suggests that it may be involved in regulating hormone action in the organism by a different mechanism than the intranuclear transcription regulation receptors. It therefore may be possible to develop drugs that have few side effects using the steroid hormone binding protein of the present invention as the target by taking advantage of the difference in binding affinities between it and the intranuclear transcription regulation type receptors with regard to steroid hormones. In particular, since hSMBP2 is derived from a human, it has clinical development advantages over proteins derived from other mammalian species such as pigs.
The steroid hormone binding protein of the present invention can be prepared either as a recombinant protein using the genetic recombination technology or as a natural protein. For example, it can be prepared as a recombinant protein by incorporating the DNA encoding the steroid hormone binding protein of the present invention (for example a DNA having the nucleotide sequence of SEQ ID NO:3) into an appropriate expression vector, introducing it into a host cell, and purifying the protein from the transformant thus obtained. It can also be prepared as a natural protein by first preparing a column using the antibody obtained from a small animal immunized with the prepared recombinant protein, then performing affinity chromatography on the extracts from the tissues or cells that highly express the steroid hormone binding protein of the present invention (for example testis or cancer cells) using the said column.
The present invention also includes proteins that are functionally equivalent to hSMBP2 protein. Here xe2x80x9cfunctionally equivalentxe2x80x9d means that the protein in question has binding activity for steroid hormones similar to the hSMBP2 protein. xe2x80x9cHaving steroid hormone-binding activityxe2x80x9d means that the protein has an activity to bind to at least one kind of steroid hormone. Steroid hormone-binding activity can be detected, for example, by testing for binding activity using commercially available tritium-labeled steroid hormones.
Proteins that are functionally equivalent to the hSMBP2 protein can be prepared by mutating the amino acid sequence of the hSMBP2 protein. As an example, one can introduce an appropriate substitution or other modification that does not affect its function into the steroid hormone binding protein of the present invention having the amino acid sequence described in SEQ ID NO.:4, resulting in the isolation of a protein that is functionally equivalent to the hSMBP2 protein. The steroid hormone binding protein of the present invention also includes proteins that are functionally equivalent to the hSMBP2 protein and that have amino acid sequences resulting from the substitution, deletion, and/or addition of one or more amino acid to the amino acid sequence described in SEQ ID NO:4.
The term xe2x80x9csubstantially purexe2x80x9d as used herein in reference to a given polypeptide means that the polypeptide is substantially free from other biological macromolecules. The substantially pure polyp pried is at least 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Purity can be measured by any appropriate standard method, for example, by column chromatography polyacrylamide gel electrophoresis, or HPLC analysis.
Methods of mutating amino acid sequences of proteins include a site-specific mutagenesis system using PCR (GIBCO-BRL, Gaithersburg, Md.) and a site-specific mutagenesis method using oligonucleotides (Kramer, W. and Fritz, H. J. (1987) Methods in Enzymol., 154: 350-367). There are no restrictions on the number of amino acids to be mutated as long as the steroid hormone-binding activity is retained. However, the number of amino acids to be mutated is usually 30 amino acids or less, preferably 20 amino acids or less, more preferably 10 amino acids or less, and still more preferably 5 amino acids or less.
A xe2x80x9cconservative amino acid substitutionxe2x80x9d is one in which an amino acid residue is replaced with another residue having a chemically similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic s de chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
It is also possible that amino acid mutations within a protein occur naturally, and the steroid hormone bindin protein of the present invention also includes such mutant proteins as produced in this manner.
Other methods o preparing proteins that are functionally equivalent to the hSMBP2 protein include using the hybridization technique. Specifically, a person skilled in the art can use a well-known hybridization technique (Sambrook, J. et al., Molecular Cloning 2nd ed. 9.47-9.58, Cold Spring Harbor Lab. Press, 1989) to isolate a DNA having a high degree of homology to the DNA sequence encoding the xe2x80x9chSMBP2xe2x80x9d protein by using the DNA sequence (SEQ ID NO:3) or a part thereof, and obtain a protein that is functionally equivalent to the xe2x80x9chSMBP2,xe2x80x9d protein by means of the said DNA. The steroid hormone binding protein of the present invention also includes proteins encoded by DNAs capable of hybridizing with DNAs comprising the DNA sequence described in SEQ ID NO:3 and that are functionally equivalent to the hSMBP2 protein.
Hybridization stringencies, as used herein, are defined as about 52xc2x0 C., 2xc3x97SSC, 0.01% SDS (low stringency); about 50xc2x0 C., 2xc3x97SSC, 0.01% SDS (medium stringency); or about 65xc2x0 C., 2xc3x97SSC, 0.01% SDS (high stringency). As the above indicates, higher temperatures are expected to yield more isolation of DNA having high homology. A person skilled in the art can realize similar stringency by considering, in addition to the temperature, such factors as the probe concentration, probe length, salt strength, and reaction time, as the hybridization stringency.
The protein encoded by the DNA obtained by means of the hybridization technique usually has a high degree of homology to the hSMBP2 protein (SEQ ID NO:4) in terms of the amino acid sequence. xe2x80x9cA high degree of homologyxe2x80x9d means 40% or more homology, preferably 60% or more homology, and more preferably 80% or more homology. The xe2x80x9cpercent identityxe2x80x9d of two amino acid sequences or of two nucleic acids is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performed with the NBLAST program, scor=100, wordlength=12. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3. Where gaps exist between two sequences, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://www.ncbi.nlm.nih. gov.
The present invention also relates to DNAs encoding the steroid hormone binding protein of the present invention. There are no particular restrictions concerning the DNA of the present invention as long as it can encode the steroid hormone binding protein of the present invention, and it includes cDNAs, genomic DNAs, and chemically synthesized DNAs. cDNAs can be prepared, for example, by preparing primers based on the nucleotide sequence described in SEQ ID NO:3 and performing RT-PCR. Genomic DNAs can be prepared by the plaque hybridization method using xcexphage. The nucleotide sequences of the DNAs thus obtained can be determined by an ordinary method using, for example, a commercially available dye terminator sequencing kit (Applied Biosystems).
An xe2x80x9cisolated nucleic acidxe2x80x9d is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. Specifically excluded from this definition are nucleic acids present in mixtures of different (i) DNA molecules, (ii) transfected cells, or (iii) cell clones: e.g., as these occur in a DNA library such as a cDNA or genomic DNA library.
The present invention also relates to vectors into which the DNA of the present invention is inserted. There are no particular restrictions concerning the vectors into which the DNA of the present invention is inserted, and a variety of vectors can be used according to the purposes. The vectors to express the steroid hormone binding protein of the present invention in vivo (for gene therapy as an example) and the vectors to prepare the recombinant protein are included. The vectors to express the steroid hormone binding protein of the present invention in vivo include the adenovirus vector xe2x80x9cpAdexLcwxe2x80x9d and the retrovirus vector xe2x80x9cpZlPneo.xe2x80x9d Expression vectors are especially useful when one wishes to use vectors to produce the steroid hormone binding protein of the present invention. Vectors such as pQE vector (Qiagen, Hilden, Germany) are preferable as the expression vectors if one wishes to use E. coli, xe2x80x9cSP-Q01xe2x80x9d (Stratagene, La Jolla, Calif.) if one wishes to use yeast, and BAC-to-BAC baculovirus expression system (GIBCO-BRL, Gaithersburg, Md.) if one wishes to use insect cells. If mammalian cells such as CHO, COS, or NIH-3T3 cells are used, vectors such as the LacSwitch II expression system (Stratagene, La Jolla, Calif.) are suitable. The DNA of the present invention can be inserted into a vector using a standard method (for example, refer to xe2x80x9cThe Qiaexpressionist handbook, Qiagen, Hilden, Germanyxe2x80x9d).
The present invention also relates to transformants retaining the DNA of the present invention in an expressible state. The transformants of the present invention include those that retain the vectors into which the DNA of the present invention is inserted and those that have the DNA of the present invention integrated into the host genome. However, they can also include all other forms as long as they expressibly retain the DNA of the present invention. There are no particular restrictions concerning the cells into which the vectors of the present invention are to be introduced. If one wishes to express the steroid hormone binding protein of the present invention in vivo, one can use desired cells as the target cells. For example, one can use E. coli, yeast, animal cells, or insect cells to produce the steroid hormone binding protein of the present invention. Vectors can be introduced into the cells, for example, by the electroporation and the calcium phosphate methods. A recombinant protein can be isolated and purified from a transformant constructed to produce the recombinant protein by a standard method such as those described in the literature xe2x80x9cThe Qiaexpressionist handbook, Qiagen, Hilden, Germany.xe2x80x9d
The present invention also relates to antibodies that bind to the steroid hormone binding protein of the present invention. There are no particular restrictions concerning the form of the antibody of the present invention, and it may include monoclonal antibodies as well as polyclonal antibodies. The antibodies also include the antisera obtained by immunizing rabbits or other small animals with the steroid hormone binding protein of the present invention, polyclonal antibodies and monoclonal antibodies of all classes, humanized antibodies by gene recombination, and human antibodies. They also include single-stranded antibodies, and parts of antibodies such as Fab fragments and F(abxe2x80x2)2 fragments.
The antibodies of the present invention can be prepared by the method in the following example. To prepare a polyclonal antibody, one could obtain sera by immunizing small animals such as rabbits with the steroid hormone binding protein of the present invention, applying the sera onto an affinity column coupled with the steroid hormone binding protein of the present invention, and obtaining the fraction that recognizes only the steroid hormone binding protein of the present invention. Next, one could prepare immunoglobulin G or M from this fraction by purification through a protein A or protein G column. To prepare a monoclonal antibody, one could immunize small animals such as mice with the steroid hormone binding protein of the present invention, remove the spleen from the mice, grind it into individual cells, fuse them with mouse myeloma cells utilizing reagents such as polyethylene glycol, and select the clones that produce the antibodies against the steroid hormone binding protein of the present invention from the fusion cells (hybridomas) thus created. Next, one could transplant the hybridomas obtained into the abdominal cavity of a mouse and recover the ascites from the mouse. By purifying the monoclonal antibody thus obtained by ammonium sulfate precipitation, a protein A or a protein G column, DEAE ion exchange chromatography, or an affinity column coupled with the steroid hormone binding protein of the present invention, one could obtain the preparation.
In addition to purifying or detecting the steroid hormone binding protein of the present invention, the antibodies of the present invention can also be used as drugs to promote or inhibit the signal transduction of the steroid hormone binding protein of the present invention. When administering the antibodies to humans as drugs, human antibodies or humanized antibodies are effective in terms of immunogenicity. Human antibodies can be prepared, for example, by immunizing a mouse whose immune system is replaced by a human system with the steroid hormone binding protein of the present invention. Humanized antibodies, on the other hand, can be prepared, for example, by the CDR graft method in which the antibody gene is cloned from the monoclonal antibody producing cells, and its antigenic determinant is grafted onto an existing human antibody.
The present invention also relates to methods for screening the compounds that bind to the steroid hormone binding protein of the present invention. The screening method of the present invention includes a process of selecting compounds that bind to the steroid hormone binding protein of the present invention by bringing a test sample into contact with the steroid hormone binding protein of the present invention. There are no particular restrictions concerning the test sample brought into contact with the steroid hormone binding protein of the present invention, and test samples include cell extracts, synthetic low molecular weight compound libraries, expression products of gene libraries, and synthetic peptide libraries.
A person skilled in the art can use the method below to isolate proteins that bind to the steroid hormone binding protein of the present invention or the genes thereof, using the protein of the present invention. Specifically, proteins that bind to the steroid hormone binding protein of the present invention or the genes thereof can be prepared by, for example, first constructing a cDNA library from the cells expected to express proteins that bind to the steroid hormone binding protein of the present invention (such as testis tissues or ovary tissues) using a phage vector (such as xcexgt11 or ZAP), expressing it on LB-agarose, fixing the expressed proteins onto a filter, purifying the steroid hormone binding protein of the present invention as a biotin-labeled protein or a fusion protein with the GST protein, letting it react with the filter above, and detecting the plaques expressing the proteins that bind to the steroid hormone binding protein of the present invention using streptavidin or an anti-GST antibody. This method is called the xe2x80x9cWest-western blotting methodxe2x80x9d (Skolnik, E. Y.; Margolis, B.; Mohammadi, M.; Lowenstein, E.; Fisher, R.; Drepps, A.; Ullrich, A.; and Schlessinger, J. (1991) Cloning of PI13 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell 65, 83-90).
It is also possible to prepare proteins or the genes thereof that bind to the steroid hormone binding protein of the present invention using the two hybrid system (MATCHMAKER Two-Hybrid System, Mammalian MATCHMAKER Two-Hybrid Assay Kit, MATCHMAKER One-Hybrid System (all from Clontech), HybriZAP Two-Hybrid Vector System (Stratagene) and the paper by Dalton, S. and Treisman, R. (1992) titled xe2x80x9cCharacterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element,xe2x80x9d Cell 68, 597-612). In this method, the steroid hormone binding protein of the present invention is expressed in yeast cells as a fusion protein with the SRF binding domain or the GAL4 binding domain. A cDNA library is next prepared from the cells expected to express proteins that bind to the steroid hormone binding protein of the present invention so that the protein is expressed as a fusion protein with the VP16 or the GAL4 transcription activation domain. The cDNA library is then introduced into the above yeast cells, and the library-derived cDNA is isolated from the positive clones. (When a protein that binds to the steroid hormone binding protein of the present invention is expressed in the yeast cell, the binding between the two proteins will activate a reporter gene, which makes it possible to confirm the positive clone.)
Furthermore, it is also possible to prepare proteins or the genes thereof that bind to the steroid hormone binding protein of the present invention. This may be done by applying the culture supernatants or the extracts of the cells expected to express proteins that bind to the steroid hormone binding protein of the present invention onto an affinity column coupled with the steroid hormone binding protein of the present invention then purifying the proteins that specifically bind to the column. It is further possible to obtain the DNA encoding the said proteins by analyzing the amino acid sequence of the protein thus obtained, synthesizing an oligo-DNA based on that information, and screening a cDNA library with the said DNA as a probe.
The method of screening for binding molecules by applying synthetic compounds, a natural product bank, or a random phage peptide display library to the fixed steroid hormone binding protein of the present invention, and the method of isolating low molecular weight compounds, proteins (or the genes thereof), or peptides that bind to the steroid hormone binding protein of the present invention, by high throughput screening using a combinatorial chemistry technique (Wrighton, N. C.; Farrell, F. X.; Chang, R.; Kashyap, A. K.; Barbone, F. P.; Mulcahy, L. S.; Johnson, D. L.; Barrett, R. W.; Jolliffe, L. K.; and Dower, W. J., Small peptides as potent mimetics of the protein hormone erythropoietin. Science (UNITED STATES) Jul. 26, 1996, 273 pp458-64; Verdine G L., The combinatorial chemistry of nature. Nature (ENGLAND) Nov. 7, 1996, 384 pp11-13; and Hogan, J. C. Jr. Directed combinatorial chemistry. Nature (ENGLAND) Nov. 7, 1996, 384 pp17-9) are also well known to a person skilled in the art. The compounds isolated through the methods above could be applied as drugs to promote or inhibit the activities of the steroid hormone binding protein of the present invention.
By performing similar screening procedures using a steroid hormone receptor of the intranuclear transcription regulation type and the steroid hormone binding protein of the present invention, it is also possible to screen for compounds that bind specifically to either of the two. In particular, one can bring test samples into contact with the steroid hormone binding protein of the present invention and with the steroid hormone receptor of the intranuclear transcription regulation type then select the compounds that bind specifically to either the steroid hormone binding protein of the present invention or the steroid hormone receptor of the intranuclear transcription regulation type. In this way, one can screen for compounds that bind specifically to either the steroid hormone binding protein of the present invention or the steroid hormone receptor of the intranuclear transcription regulation type. The known steroid hormone receptors of the intranuclear transcription regulation type used for this screening include progesterone receptor (Acc. 189935), glucocorticoid receptor (Acc. 121069), androgen receptor (Acc. 105325), vitamin D receptor (Acc. 340203), mineral corticoid receptor (Acc. 126885), estrogen receptor (Acc. 544257), steroid hormone receptor ERR1(Acc. 119560), and steroid hormone receptor ERR2 (Acc. 119561). These compounds can be used to suppress the side effects of the known steroid type drugs.
A compound isolated by the screening method of the present invention for use as a medicine can be formulated by means of known pharmaceutical formulation manufacturing methods. For example, the compound is administered to the patient using pharmaceutically acceptable carriers or media (isotonic sodium chloride solutions, vegetable oils, suspending agents, surfactants, stabilizers, etc.). Administration can be made percutaneously, intranasally, perbronchially, intramuscularly, intravenously, or perorally, depending on the characteristics of the compound. If the isolated compound is a DNA, it can be administered to the humans by utilizing the vectors for in vivo expression described above.
The present invention also relates to DNAs having a chain length of at least 15 nucleotides that hybridize specifically with DNAs comprising the nucleotide sequence described in SEQ ID NO:3 (including its complementary strand). Here, xe2x80x9chybridize specificallyxe2x80x9d means that there is no significant cross-hybridization with DNAs encoding other proteins under standard hybridization conditions, and preferably under stringent hybridization conditions. Such DNAs can be used as probes to detect or isolate the DNA encoding the proteins of the present invention and as primers to amplify the same. A person skilled in the art can select the appropriate stringency needed for the specific hybridization by considering such parameters as hybridization temperature, reaction time, probe or primer concentration, probe or primer length, and salt strength.
Furthermore, the xe2x80x9cDNAs comprising at least 15 nucleotides that hybridize specifically with DNAs comprising the nucleotide sequence described in SEQ ID NO:3xe2x80x9d of the present invention include, for example, antisense oligonucleotides and ribozymes. Antisense oligonucleotides act on the cells that produce the protein of the present invention, bind to the DNA or mRNA encoding the said protein, and suppress the protein""s action through suppressing the expression of the protein of the present invention by inhibiting the transcription or translation, or by promoting the mRNA degradation. The antisense oligonucleotides include those that hybridize with the nucleotide sequence described in SEQ ID NO:3 at some positions. The antisense oligonucleotides are preferably antisense oligonucleotides against 15 or more consecutive nucleotides within the nucleotide sequence described in SEQ ID NO:3. More preferably, the antisense oligonucleotides are the above antisense oligonucleotides in which the above 15 or more consecutive nucleotides include a translation initiation codon.
The antisense oligonucleotides can be derivatives or modifications. These modifications include, for example, lower alkylphosphonate modifications such as the methylphosphonate type or the ethylphosphonate type, phosphorothioate modifications, and phosphoramidate modifications.
The antisense oligonucleotides include not only those that are completely complementary to the corresponding nucleotides constituting the given region of the DNA or mRNA, but also those that may contain one or more mismatches, as long as the oligonucleotides can selectively and stably hybridize with the DNA or mRNA of the nucleotide sequence described in SEQ ID NO:3. Such a DNA has at least 70%, preferably at least 80%, more preferably at least 90%, and still more preferably at least 95% nucleotide sequence homology in the region of at least a 15 consecutive nucleotide sequence. It is worth noting that the algorithm described in the literature cited above can be used to determine the homology.
The antisense oligonucleotides of the present invention can be made into external preparations such as paints and poultices by mixing with an appropriate base that is inactive toward the oligonucleotides. They can also be made into tablets, powders, granules, capsules, liposome capsules, injections, liquid preparations, or nasal drops by adding, if necessary, excipients, isotonizing agents, solubility increasing agents, stabilizers, antiseptics, and analgesic agents, or further made into freeze-dried preparations. These can be prepared according to standard methods.